The serotonin transporter is responsible for terminating the action of serotonin that has been released into the synaptic cleft. The activity of this transmembrane protein is blocked by antidepressant drugs and cocaine, and modulated by amphetamine derivatives, some of which are neurotoxic. The mechanism by which the transporter accumulates serotonin within the cell is understood in broad terms. The energy for transport comes from Na+, C1 and K= ions flowing downhill across the plasma membrane. Now that the primary structure of the serotonin transporter is known from cDNA cloning, it is important to understand how the various functions of the transporter, including its interactions with drugs of abuse, are related tot he sequence and ultimately to the structure. This application proposes two approaches to this problem. The first approach will yield information on the transmembrane orientation and functional importance of hydrophilic "loops" believed to connect transmembrane domains. Mutated forms of the transporter containing unique tagged sequence elements will be expressed in mammalian cells and analyzed biochemically and physiologically. The accessibility of various parts of the transporter protein to the cell surface will be determined using a variety of membrane-impermeant reagents. At the same time the transport properties and drug sensitivities of the tagged mutants will be tested. In addition to providing a better picture of the transmembrane topology, this approach will address basic questions of transporter dynamics, and help to localize drug binding sites on the transporter surface. The second approach will examine the effects of stably expressing the serotonin transporter at high levels in different cell types. These studies will use inducible promoters to test the hypothesis that expression of the transporter is inhibitory to cell growth. In particular, the ability of amphetamines to stimulate transporter activity, and consequently dissipate transmembrane ion gradients, will be addressed. These studies are expected to shed light on the ways that the serotonin transporter mediates degeneration of nerve terminals in the presence of amphetamine derivatives.